Toxicology in the 21st Century (Tox21)

Robotic arm used for Tox21 testing

Thousands of chemical substances exist in the world, but only a small fraction of these have been adequately assessed for their potential toxicity to humans. The Toxicology in the 21st Century program, or Tox21, is a unique collaboration between several federal agencies to develop new ways to rapidly test whether substances adversely affect human health. Substances assayed in Tox21 include a diverse range of products such as: commercial chemicals, pesticides, food additives/contaminants, and medical compounds.

The following four government agencies bring their unique expertise, resources, and tools to the Tox21 collaboration:

  • National Institute of Environmental Health Sciences (NIEHS) / National Toxicology Program (NTP),
    National Institutes of Health (NIH), U.S. Department of Health and Human Services
  • National Center for Advancing Translational Sciences (NCATS),
    National Institutes of Health (NIH)
  • U.S. Food and Drug Administration (FDA),
    U.S. Department of Health and Human Services
  • National Center for Computational Toxicology,
    Office of Research and Development, U.S. Environmental Protection Agency (EPA)

Goals of the Tox21 Program

The goal of Tox21 is to research, develop, evaluate, and translate innovative test methods that will better predict how substances may affect humans and the environment. This includes the use of robotics in high-throughput screening (HTS) in Tox21 as well as the use of other new approach methodologies to evaluate substance toxicity.

The use of robotics in HTS is important because of the ability to increase the volume and speed of testing. While many conventional pre-clinical tests use animals, these approaches are often slow and expensive. In addition, they provide no guarantee of predicting toxic health effects on humans later observed in clinical trial settings. Automated, "high-throughput" screening serves an important role as an alternative method for animal use, as well as an efficient and forward-thinking use of scientific resources.

Tox21 aims to:

  • Prioritize substances for further in-depth toxicological evaluation
  • Identify mechanisms of action for further investigation (e.g., toxicity-associated and disease-associated pathways)
  • Develop models that better predict how substances will affect biological responses (predictive toxicology)
  • Employ testing methods using human cells (in vitro approaches)
  • Reduce time, effort, and costs associated with testing
  • Contribute to the reduction, refinement, and replacement of animals used in toxicity testing

Research Phases

Since 2008, NIEHS and collaborating agencies have created a three-phase plan. Today, Tox21 is in the Phase III stage of the collaboration.

Phase I & II

Some milestones achieved during Phase I and Phase II:

Phase Milestones
Phase I
  • Demonstrate robotic testing could produce high-quality test results for thousands of substances, with the goal of reduction in animal testing.
  • By 2010, the use of robotics for high-throughput screening yielded high quality data on environmental substances in a fraction of the time required with traditional animal testing.
Phase II
  • The quantity of compounds tested and types of assays (methods or testing) used were greatly increased.
  • The chemical library was expanded to more than 10,000 compounds, including industrial and consumer products, food additives, drugs, and chemical mixtures.
  • Tox21 partners are pursuing additional cell-based experiments to further define and clarify chemical exposure effects.

Phase III

In Phase III, the Tox21 federal partners developed a document entitled "The US Federal Tox21 Program: A strategic and operational plan for continued leadership" that was recently published (Thomas et al., ALTEX 2018 Mar 8. DOI: 10.14573/altex.1803011.). In this document, five key Areas of Focus were identified for moving forward in Phase III, which were:

  • Develop alternative test systems that are predictive of human toxicity and dose response
  • Address key technical limitations of current in vitro test systems
  • Curate and characterize legacy in vivo toxicity studies
  • Establish scientific confidence in in vitro test systems and integrated assay batteries
  • Refine and deploy in vitro methods for characterizing pharmacokinetics and in vitro disposition

In one Phase III project, Tox21 partners have identified approximately 2,800 human genes that can serve as "Sentinel" genes (the S1500+ gene set) to interrogate responses in cells and tissues to toxic substances. Identification of similar gene sets for rats, mice, and zebrafish are in process. A new test using these genes will study changes in levels of ribonucleic acid (RNA) messages (or transcripts), which may ultimately reveal the effect of substances on our genes.

Phase III will also attempt to predict human disease by assessing the effects of substances on cells more like those in human organs, such as the liver, heart, or brain. Phase III will test substances in 3-D organ-like models, as well as organisms, like zebrafish, that have shared biological complexity with humans.

Scientists can already predict toxicity, if a substance's structure is similar to that of a known toxicant, using a process referred to as "read across." Ultimately, researchers hope to create better computational models, including biological response read across approaches, to better predict whether a substance might be toxic to humans.

More details on Tox21 and its testing phases are available in the Tox21 Fact Sheet.


For further information on NTP's Tox21 program, contact:

  • David M. Reif, Ph.D.
  • Chief, Predictive Toxicology Branch
  • 530 Davis Dr Durham, NC 27713
  • 984-287-3195